To study the novel bacterial-host cell interactions associated with intracellular infection by Legionella pneumophila , we developed a new transposon mutagenesis system. Mini-Tn10phoA generates translational gene fusions with the E. coli alkaline phosphatase gene, permitting us to identify insertions in genes encoding signal sequences. We generated a panel of 170 PhoA+ mutants and identified 23 that are unable to produce cytopathicity of U937 (macrophage-like) cells or to replicate in the amoebae, Hartmannella vermiformis. My hypothesis is that these avirulent mutants will have defects in intracellular trafficking, i.e., delivery to the lysosome or other non-permissive compartment. I am using immunofluorescence microscopy with antibodies directed at organelle-specific markers to determine the intracellular fate of these mutants. Ultimately, I expect to isolate bacterial factors that influence intracellular trafficking.